Project Summary/Abstract The goal of this project is to obtain detailed understanding of the structural features present in a class of multinuclear metalloenzymes that make it capable of promoting selective 6 e- reduction of sulfite (SiRs), and address important scientific issues in the fields of complex multinuclear transition metal active sites and the global sulfur cycle. Specifically, this project seeks to understand the role of the coupled auxiliary [4Fe-4S] cluster reduction potential on facilitating efficient?and selective?S-O bond cleavage of sulfite, along with the chemical nature of intermediates that have been proposed in the catalytic cycle of SiRs. To achieve this goal, this project proposes complimentary studies, both of a native SiR protein, and a biosynthetic model capable of catalyzing sulfite reduction?the only current structural and functional model of SiRs. Using a novel biosynthetic approach, which aims to overcome limitations inherent to studies of the native enzyme alone, a robust scaffold will be used to learn which features of SiR are crucial for its activity, through experiments designed to display a gain in function, as opposed to inferring structure-function relationship through loss of activity in the native enzyme. The proposed complimentary studies of these two systems will allow us to identify the structural features unique to SiRs that lead to high catalytic efficiency towards a complex multi-electron, multi-proton transformation. We will be able to understand: (1) the importance of the redox active [4Fe-4S] cluster coupled to the siroheme substrate binding site on SiR activity, (2) the nature of postulated Fe-SOx intermediates, and their relevance to the catalytic cycle of SiR, and (3) the features of the siroheme ligand in SiR which make it suitable for sulfite reduction activity, relative to the heme c of the biosynthetic scaffold. Achieving the above goals will result in a deeper understanding of the structure and function of SiRs that may be difficult to achieve by studying the native enzyme alone. By mimicking the function of a native system through rational modifications with a different protein scaffold, generalizable conclusions can be made concerning important structural features of the native active site. This research plan will advance the knowledge of a broad range of multinuclear metalloenzymes relevant to human health, specifically, related to their structure, function, and metalloenzyme design in general, while providing an excellent training opportunity to achieve the career goal the candidate.